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Highly Stable and Sensitive Nucleic Acid Amplification and Cell-Phone-Based Readout

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†Department of Bioengineering, ‡Department of Electrical Engineering, ∥Department of Pathology & Laboratory Medicine, ^Department of Mechanical and Aerospace Engineering, ⊥California NanoSystems Institute, and Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California 90095, United States
*E-mail: [email protected]
Cite this: ACS Nano 2017, 11, 3, 2934–2943
Publication Date (Web):February 24, 2017
https://doi.org/10.1021/acsnano.6b08274
Copyright © 2017 American Chemical Society
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Abstract

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Key challenges with point-of-care (POC) nucleic acid tests include achieving a low-cost, portable form factor, and stable readout, while also retaining the same robust standards of benchtop lab-based tests. We addressed two crucial aspects of this problem, identifying a chemical additive, hydroxynaphthol blue, that both stabilizes and significantly enhances intercalator-based fluorescence readout of nucleic acid concentration, and developing a cost-effective fiber-optic bundle-based fluorescence microplate reader integrated onto a mobile phone. Using loop-mediated isothermal amplification on lambda DNA we achieve a 69-fold increase in signal above background, 20-fold higher than the gold standard, yielding an overall limit of detection of 25 copies/μL within an hour using our mobile-phone-based platform. Critical for a point-of-care system, we achieve a >60% increase in fluorescence stability as a function of temperature and time, obviating the need for manual baseline correction or secondary calibration dyes. This field-portable and cost-effective mobile-phone-based nucleic acid amplification and readout platform is broadly applicable to other real-time nucleic acid amplification tests by similarly modulating intercalating dye performance and is compatible with any fluorescence-based assay that can be run in a 96-well microplate format, making it especially valuable for POC and resource-limited settings.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b08274.

  • Real-time fluorescence measurements of additional EvaGreen concentrations; real-time fluorescence measurements of SYBR Green and acridine orange with and without HNB; scheme of image processing and device calibration; limit of detection studies of the mobile phone fluorescence reader (PDF)

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